Measured pulmonary arterial tissue stiffness is highly sensitive to AFM indenter dimensions

Abstract

The mechanical properties of pulmonary tissues are important in normal function and the development of diseases such as pulmonary arterial hypertension. Hence it is critical to measure lung tissue micromechanical properties as accurately as possible in order to gain insight into the normal and pathological range of tissue stiffness associated with development, aging and disease processes. In this study, we used atomic force microscopy (AFM) micro-indentation to characterize the Young's modulus of small human pulmonary arteries (vessel diameter less than 100µm), and examined the influence of AFM tip geometry and diameter, lung tissue section thickness and the range of working force applied to the sample on the measured modulus. We observed a significant increase of the measured Young's modulus of pulmonary vessels (one order of magnitude) associated with the use of a pyramidal sharp AFM tips (20nm radius), compared to two larger spherical tips (1 and 2.5µm radius) which generated statistically indistinguishable results. The effect of tissue section thickness (ranging from 10 to 50 μm) on the measured elastic modulus was relatively smaller (<1-fold), but resulted in a significant increase in measured elastic modulus for the thinnest sections (10 μm) relative to the thicker (20 and 50 μm) sections. We also found that the measured elastic modulus depends modestly (again <1-fold), but significantly, on the magnitude of force applied, but only on thick (50 μm) and not thin (10 μm) tissue sections. Taken together these results demonstrate a dominant effect of indenter shape/radius on the measured elastic modulus of pulmonary arterial tissues, with lesser effects of tissue thickness and applied force. The results of this study highlight the importance of AFM parameter selection for accurate characterization of pulmonary arterial tissue mechanical properties, and allow for comparison of literature values for lung vessel tissue mechanical properties measured by AFM across a range of indenter and indentation parameters.

Keywords: AFM; Elasticity; Lung tissue; Micro-indentation; Pulmonary artery; Young's modulus.

Figure 1

Bright field images (X200) of human lung tissue cut at (A) 10, (B) 20 and (C) 50 μm section thicknesses and mounted on glass slides. Scale bar = 100 μm. Presence of one pulmonary artery in the lung section. In the inset of (A) is shown an optical image of an AFM tip indenting lung tissue. White crosses indicate the location of the 5 points of measurement onto the vessel wall. Scale bar = 50 μm.

Figure 2

(A) Elastic modulus of human pulmonary vessels estimated by AFM indentation performed with three different AFM tips: sharp pyramidal tip at 20 nm radius (half-open angle of 18°), spherical tip at 1 μm radius and spherical tip with a radius of 2.5 μm. Force curves were performed on 4 vessels from 4 different lung donors (one vessel per donor). (B) Mean ± SD of elastic modulus determined by fitting force curves with Sneddon’s (for pyramidal AFM tip) and Hertz’s (for spherical AFM tip) models. Contact radii a and areas S_c were calculated from the equations (4) and (5) for spherical tips and (7) and (8) for pyramidal tip with the mean value of the indentation depth δ. (C) Elastic modulus extracted from force curves performed with three different AFM tips on human pulmonary arteries, plotted as a function of the indentation depth. The size of the AFM tips is indicated to compare with the indentation depth values range. For the pyramidal tip, the tip height is estimated as 6 μm. (D) Elastic modulus estimated by fitting force curves considering large (entire curve) or limited (δ ≤ 500 nm) indentation depths. Analysis shown for all curves from one sample (from Table 2). Comparison of mean values for each tip was conducted using a Mann-Whitney test for each of the three AFM tips.

Figure 3

(A) Representative force curves (force - z displacement) obtained on pulmonary arteries at different lung tissue thicknesses. (B) Dispersion of elastic values for 10 force curves taken at the same location. (C) Elastic modulus of human pulmonary arteries from force curves performed on lung tissue sections cut at 10, 20 and 50 μm thicknesses. (D) Mean ± SD of Young’s modulus of micro vessels. Mean ± SD of indentation depth calculated from equation (1) for all the force curves.

Figure 4

(A) Elasticity of pulmonary vessels measured by AFM indentation. Correction factor developed by Dimitriadis et al. (2002) applied on the experimental results. Mean ± SD are presented in (B). Lung tissue was considered bonded to the glass slide with no adhesion between the tip and the sample.

Figure 5

Elastic modulus of pulmonary arteries from force curves performed at different working forces. Lung tissue sections cut at 10 μm (A) and 50 μm (B) thicknesses. E values at 97% were used as a reference for statistical comparisons by ANOVA (p < 0.05).